Retroviral vectors derived from murine leukemia virus (MLV) have been employed in more than 50% of approved clinical gene therapy trials (Wiley—The Journal of Gene Medicine Website. However, one of the major limiting factors hindering a wider use of these vectors is that the level of gene expression is does not get high enough to give clear therapeutic effects. The present inventors previously constructed retroviral vectors that contains no viral coding sequence and harbors a heterologous splicing acceptor sequence from cellular or other viral genes (KR Patent Laid-Open Publication No. 2000-6334). One of the vectors contains a splicing acceptor from the human EF1α gene. This vector gives a significantly higher level of gene expression than the control vector lacking such a splicing acceptor sequence. However one problem with this vector was that viral titer varied depending on the packaging lines used. For example, when the NIH3T3-based PG13 line was used, viral titer decreased about 10 folds. In FLYA13 derived from HT1080 cells, there was a 3-fold decrease in viral titer. Results from RNA analysis indicated that low viral titer is due to highly efficient splicing of the genomic size transcript containing the packaging signal sequence.
The present invention relates to further improvement of this retroviral vector by introducing mutations into the region around the splicing acceptor. Introduction of these mutations make a retroviral vector produce viral titer close to that of the control, while still producing high levels of gene expression. Therefore, this vector should be much more effective than others in retroviral gene therapy.